Persistent infections with Human enteroviruses (HEV) are a major cause of dilated cardiomyopathies, and have been causally implicated in type 1 diabetes, gastrointestinal and nervous system disorders. HEVs are acid-resistant, and can survive in the gastrointestinal tract and other organs for long periods of time. There are currentl no drugs approved to treat HEV infections. The goal of this high-risk/high-reward project is to identify inhibitors of uridylylation of a small protein, VPg, and show that these compounds provide an alternative treatment route for treating chronic infections with slow replicating HEV strains. As uridylylation is virus-specific, the inhibitors should have fewer side effects on mammalian cells. This translational work will combine computational, structural and high throughput screening (HTS) methods to select inhibitors that interfere with uridylylation, and HEV replication. Uridylylation inhibitors selected, in the R21 phase, will be tested, in the R33 phase, for their effects on inhibiting HEV in mammalian cells and in a murine model of chronic HEV infection. Aim 1 is to develop uridylylation assays using a consensus VPg, and screen a diversity set of compounds for those that interfere with this reaction. Active compounds identified in the HTS will be characterized in Aim 2 to derive structure-activity relationships and identify substructures for screening large, web-based compound libraries. A pharmacophore based on VPg structures will be used for further in silico screening of these libraries. Selected compounds will be purchased and assayed, or produced in-house. Assuming that 3-10 uridylylation inhibitors have been identified, the R33 phase of the work will begin. Aim 3 is to further characterize the activity of the uridylylation inhibitors and select those most suitable fo biological assays according to their pharmaceutical properties (low toxicity, good solubility and high stability in cell culture media). Compounds may be formulated in nanoparticles to enhance cell uptake. The most potent, biologically acceptable inhibitors will be further tested for their ability to diminish the replication of HEV strains in infected cells, alone or in combination with other active compounds and antiviral agents. Solution studies using NMR and native gel electrophoresis, and co-crystal structures of the polymerase with the best antiviral compounds will be used to establish their mode of activity. Finally, the antivirals will be tested for their bility to halt HEV replication in cultured cardiomyocytes and a murine model of chronic infection with Coxsackie virus B3. Successful inhibitors will be potential new therapies for HEV infections, that can be used alone or in combination with other compounds developed as part of the Global Polio Eradication Initiative, including capsid binding compounds that inhibit cell entry. Anti-HEV compounds that target uridylylation may thus also play a role in securing polio eradication.